DNA疫苗的不同构象在体内外表达差异研究
Study on differences in expression levels in vitro and in vivo between different conformational forms of DNA vaccine
分类号:
出版年·卷·期(页码):2013,33 (8):0-0
DOI:
10.16155/j.0254-1793.2017.01.01
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目的:研究DNA疫苗构象对体内和体外表达的影响。方法: 分别构建绿色荧光蛋白表达质粒(pcDNA3.1-EGFP)和萤火虫荧光素酶表达质粒(pDRVISV1.0-Fluc),对其存在的3种拓扑形式(包括超螺旋、线性、闭环切刻等)和反复冻融状态对表达效率的影响进行分析,前者用脂质体介导转染293FT细胞,用流式细胞仪检测EGFP的表达情况;后者通过接种BALB/c小鼠,用活体成像技术监测荧光素酶的表达情况。结果: 对pcDNA3.1-EGFP质粒,流式细胞仪检测其转染293FT细胞48 h后显示,4种处理组的质粒表达EGFP的效率和荧光强度有所差异,依次为新鲜制备质粒(89.76%,2682.68)>反复冻融处理质粒(76.35%,2313.48)>闭环切刻质粒(68.45%,1245.95)>线性质粒(38.28%,929.5)。对pDRVISV1.0-Fluc质粒,采用小鼠活体成像技术持续观察接种后28 d的体内荧光素酶的荧光信号强度情况,结果显示:接种后0.2 d观察到荧光,之后强度持续开始增强,至第5 d达到平台期,维持至18 d。质粒各处理组的荧光强度组间有显著差异,依次为新鲜制备质粒(108.6)>反复冻融处理质粒(108.4)>线性质粒(108.3)>切刻质粒(108.0)。2种质粒不同构象的体内体外表达中,均是新鲜制备质粒的表达量最高,因其中超螺旋为其主要构象。结论: 质粒中超螺旋比例高时其转染效率及表达量最高,提高超螺旋比例是DNA疫苗质量控制的关键。
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1 Prazeres DM,Ferreira GN,Monteiro GA,et al.Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy:problems and bottlenecks.Trends Biotechnol,1999,17(4):169
2 Eastman EM,Durland RH.Manufacturing and quality control of plasmid-based gene expression systems.Adv Drug Deliv Rev,1998,30(1-3):33
3 Middaugh CR,Evans RK,Montgomery DL,et al.Analysis of plasmid DNA from a pharmaceutical perspective.J Pharm Sci,1998,87(2):130
4 Stadler J,Lemmens R,Nyhammar T.Plasmid DNA purification.J Gene Med, 2004,6(Suppl 1):S54
5 Sorensen SJ,Sorensen AH,Hansen LH,et al. Direct detection and quantification of horizontal gene transfer by using flow cytometry and gfp as a reporter gene.Curr Microbiol,2003,47(2):129
6 Zhang WS,Purchio AF,Chen K,et al.A transgenic mouse model with a luciferase reporter for studying in vivo transcriptional regulation of the human CYP3A4 gene.Drug Metab Dispos, 2003,31(8):1054
7 Bremer C,Weissleder R.In vivo imaging of gene expression.Acad Radiol,2001,8(1):15
8 Chiarella P,Fazio VM,Signori E.Application of electroporation in DNA vaccination protocols.Curr Gene Ther,2010,10(4):281
9 van Drunen Littel-van den Hurk S,Hannaman D.Electroporation for DNA immunization:clinical application. Expert Rev Vaccines, 2010,9(5):503
10 Guidance for Industry:Considerations for Plasmid DNA Vaccines for Infectious Disease Indications,US Food and Drug Administration. .http://www.fda.gov/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/vaccines/ucm074770.htm
11 SFDA.Technical Guideline for Pre-Clinical Research of Preventive DNA Vaccines (预防用DNA疫苗临床前研究技术指导原则).2003
12 Pillai VB,Hellerstein M,Yu T, et al.Comparative studies on in vitro expression and in vivo immunogenicity of supercoiled and open circular forms of plasmid DNA vaccines.Vaccine,2008,26(8):1136
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